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A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium
supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l−1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog
medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l−1 timentin, and 30 mg l−1 kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase
chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested.
The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence
of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization
of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black
cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry
genotypes. 相似文献
3.
A. Karthikeyan J. Shilpha S. Karutha Pandian M. Ramesh 《Plant Cell, Tissue and Organ Culture》2012,109(1):153-165
A reproducible and highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of indica rice (Oryza sativa L. subsp. indica cv. ADT 43) was established. Prior to transformation, embryogenic callus were induced from mature seeds incubated on Linsmaier
and Skoog (LS) medium supplemented with 2.5 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l−1 thiamine-HCl. Callus, intact mature seeds, and other in vitro derived explants (leaf bases, leaf blades, coleoptiles, and
root-tips) were immersed in a bacterial suspension culture of A. tumefaciens strain EHA 105, OD600 of 0.8, and co-cultivated on LS medium for 2 days in the dark at 25 ± 2°C. Based on GUS expression
analysis, 10 min incubation time of explants on a co-cultivation medium containing 100 μM acetosyringone was optimum. Following
β-glucuronidase (GUS) assay and polymerase chain reaction (PCR) analysis, transformants were identified. Stable integration
of the transgene was confirmed in four putatively transformed T0 plants by Southern blot analysis. The copy number of the transgene in these lines, one to two, was then determined. Among
the observations made, necrosis of co-cultivated explants was a problem, as well as sensitivity of callus to Agrobacterium infection. Levels of necrosis could be minimized following co-cultivation of explants in a medium consisting of 30% LS and
containing 10 g l−1 (14), polyvinyl pyrrolidone, 10% coconut water, and 250 mg l−1 timentin (15:1). This latter medium also increased the final transformation efficiency to 15.33%. 相似文献
4.
A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from
Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166
and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM
2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin
(Km) were then recovered on CIM + 25 mg l−1 Km + 250 mg l−1 timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per
explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM
α-naphthaleneacetic acid (NAA) + 25 mg l−1 Km + 250 mg l−1 timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and
18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration
of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot
analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production
of transgenic celery plants. 相似文献
5.
A genetic transformation system has been developed for callus cells of Crataegus
aronia using Agrobacterium
tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with
5 mg l−1 Indole-3-butyric acid (IBA) and 0.5 mg l−1 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different
types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red
colored callus was obtained on MS medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l−1 kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli
were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l−1 hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this
is the first time to report an Agrobacterium-mediated transformation system in Crataegus
aronia. 相似文献
6.
7.
N. Selvaraj S. Kasthurirengan A. Vasudevan M. Manickavasagam C. W. Choi A. Ganapathi 《In vitro cellular & developmental biology. Plant》2010,46(4):329-337
Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis
from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II (nptII) and phosphinothricin resistance genes (bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence
of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, 1962) medium supplemented with 1 mg L−1 benzyladenine (BA), 20 mg L−1
l-glutamine and 2 mg L−1 phosphinothricin (PPT) or 100 mg L−1 kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at
regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants
were achieved on PPT (2 mg L−1) containing MS media with 0.5 mg L−1 gibberellic acid (GA3) and 0.6 mg L−1 indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T0 and the progenies. T1 segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation
efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with
effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher
number of transgenic cucumber plants. 相似文献
8.
F. D. Espasandin M. M. Collavino C. V. Luna R. C. Paz J. R. Tarragó O. A. Ruiz L. A. Mroginski P. A. Sansberro 《Plant Cell, Tissue and Organ Culture》2010,102(2):181-189
A protocol for the production of transgenic plants was developed for Lotus tenuis via Agrobacterium-mediated transformation of leaf segments. The explants were co-cultivated (for 3 days) with an A. tumefaciens strain harbouring either the binary vector pBi RD29A:oat arginine decarboxylase (ADC) or pBi RD29A:glucuronidase (GUS), which
carries the neomycin phosphotransferase II (nptII) gene in the T-DNA region. Following co-cultivation, the explants were cultured in Murashige and Skoog medium supplemented
with naphthalenacetic acid (NAA) and benzyladenine (BA) and containing kanamycin (30 μg ml−1) and cefotaxime (400 μg ml−1) for 45 days. The explants were subcultured several times (at 2-week intervals) to maintain the selection pressure during
the entire period. About 40% of the explants inoculated with the pBiRD29:ADC strain produced eight to ten adventitious shoots
per responsive explant through a direct system of regeneration, whereas 69% of the explants inoculated with the pBi RD29A:GUS
strain produced 13–15 adventitious shoots per responsive explant. The selected transgenic lines were identified by PCR and
Southern blot analysis. Three ADC transgenic lines were obtained from 30 infected explants, whereas 29 GUS transgenic lines
were obtained from 160 explants, corresponding to a transformation efficiency of 10 and 18.1%, respectively. More than 90%
of the in vitro plantlets were successfully transferred to the soil. The increase in the activity of arginine decarboxylase
from stressed ADC- Lt19 lines was accompanied by a significant rise in the putrescine level. The GUS transgenic line driven by the RD29A promoter
showed strong signals of osmotic stress in the leaves and stem tissues. All of the transgenic plants obtained exhibited the
same phenotype as the untransformed controls under non-stress conditions, and the stability of the gene introduced into the
cloned materials was established. 相似文献
9.
Eline Kirk Mørk Karin Henriksen Henrik Brinch-Pedersen Kell Kristiansen Karen Koefoed Petersen 《Plant Cell, Tissue and Organ Culture》2012,108(3):501-512
A protocol for adventitious shoot formation in Symphyotrichum novi-belgii was developed after investigating the effects of cultivar and hormone combinations. A Murashige and Skoog medium with 1.0 mg l−1 6-benzyladenine induced adventitious shoot formation in 15 out of 19 cultivars. Addition of 0.1 mg l−1 indole-3-acetic acid or naphthaleneacetic acid increased the total number of shoots per explant, but not the number of shoots
longer than 1 cm. Addition of dichlorophenoxyacetic acid (2,4-D) promoted callus formation, but inhibited shoot elongation.
A transformation system for the two cultivars Victoria Fanny and Victoria Jane was developed by co-cultivation of leaf explants
with Agrobacterium tumefaciens. Three bacterial strains (LBA 4404, A281 and C58) all carrying the binary vector, p35S-GUS-INT, and harbouring the uidA gene coding for β-glucuronidase (GUS) were used. Regeneration of transgenic plants after co-cultivation with A281 was independent
of cultivar, and all explants produced callus followed by indirect shoot formation. In ‘Victoria Fanny’ shoots were formed
faster and without a callus phase after co-cultivation with LBA 4404 or C58. The highest number of potentially transformed
shoots was regenerated after co-cultivation of ‘Victoria Fanny’ leaf explants with LBA 4404. Integration of the transgenes
in the plant genome was confirmed using PCR and Southern blot hybridisation. To verify that the transgenes could be transferred
to offspring, crosses were conducted between three transgenic lines of ‘Victoria Fanny’ and two wild type pollen donors. It
was demonstrated that viable seeds were produced and that the uidA gene was inherited. 相似文献
10.
Trifolium alexandrinum L. (Egyptian clover) is one of the most important forage crops in the world. Its regeneration in tissue culture has been
described in a few reports but the efficiency, accurate time scales and applicability to various genotypes of the described
procedures are uncertain. Therefore their suitability for genetic transformation is unclear. In this study, were report new
fast procedures for regeneration of Egyptian clover that are applicable to the regeneration of various genotypes (Mescawi-ahaly,
Sakha3 and Sakha4). Shoots were regenerated from intact and wounded cotyledons as well as hypocotyls of Mescawi-ahaly on naphthaleneacetic
acid/benzyladenine (NAA/BA) and naphthaleneacetic acid/thidiazuron (NAA/TDZ) media. The highest shoot regeneration frequencies
were obtained from intact cotyledons on NAA/BA (0.05 mg l−1 NAA combined with 2.0 mg l−1 BA) and NAA/TDZ (0.05 mg l−1 NAA combined with 1.0 mg l−1 TDZ) media (66.2 and 43.1% respectively) compared to 18.4 and 10.1% for wounded cotyledons on NAA/BA and NAA/TDZ respectively.
21.0% shoot regeneration frequency was observed for hypocotyls on NAA/BA (2.0 mg l−1 NAA combined with 0.5 mg l−1 BA) medium but no regeneration was obtained on NAA/TDZ medium. Rooting of the regenerated shoots was induced on indole butyric
acid (IBA: 0.24 mg l−1) or NAA (2.0 mg l−1) media where IBA medium supported significantly higher frequencies of rooting as well as survival of the whole plantlets
after transfer to soil. However, the rooting and survival frequencies also depended on the type of explant and the medium
used for shoot regeneration. The two cultivars Sakha3 and Sakha4 were regenerated using the culture conditions optimized for
Mescawi-ahaly with comparable efficiencies, indicating that the described procedure is not genotype dependent. The time scale
of whole plantlet regeneration ranged from 7.5 weeks for intact and wounded cotyledons to 10 weeks for hypocotyl explants. 相似文献
11.
Meiru Li Hongqing Li Xiaoying Hu Xiaoping Pan Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2010,102(3):321-327
Zoysia tenuifolia Willd. ex Trin. is one of the most popularly cultivated turfgrass. This is the first report of successful plant regeneration
and genetic transformation protocols for Z. tenuifolia using Agrobacterium tumefaciens. Initial calli was induced from stem nodes incubated on a Murashige and Skoog (1962) (MS) medium supplemented with 2 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg l−1 6-benzyladenine (BA), with a frequency of 53%. Compact calli were selected and subcultured monthly on the fresh medium. Sixty-nine
percent of the calli could be induced to regenerate plantlets when the calli incubated on a MS medium supplemented with 0.2 mg l−1 BA under darkness. For genetic transformation, calli were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. Following co-cultivation, about 12% of the callus explants produced hygromycin resistant calli on
MS medium supplemented with 2 mg l−1 2,4-D, 1 mg l−1 BA, 50 mg l−1 hygromycin, 500 mg l−1 cefotaxime after 8 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing
0.2 mg l−1 BA, 50 mg l−1 hygromycin, and 250 mg l−1 cefotaxime, and about 46% of the resistant calli differentiated into shoots. Finally, all the resistant shoots were rooted
on 1/2 MS media supplemented with 50 mg l−1 hygromycin, 250 mg l−1 cefotaxime. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. About 5% of the total
inoculated callus explants produced transgenic plants after approximately 5 months. The procedure described will be useful
for both, the introduction of desired genes into Z. tenuifolia and the molecular analysis of gene function. 相似文献
12.
In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 μM
6-benzylaminopurine (BA), 0.45 μM thidiazuron (TDZ), and 500 mg l−1 casein hydrolysate (CH) with a regeneration frequency of 64.7% after 3 wk. Subculturing explants (after harvesting shoots)
to fresh treatment medium significantly increased shoot bud regeneration (16.6 buds per explant), but the buds failed to develop
into shoots. A higher percentage (73.3%) of the explants regenerated four shoots per explant on woody plant medium (WPM) supplemented
with 4.4 μM BA, 0.29 μM gibberellic acid (GA3), and 500 mg l−1 CH after 3 wk. Explants subcultured to fresh treatment medium after harvesting shoots significantly increased shoot regeneration
(16 shoots per explant). Shoot elongation was achieved (4 cm) when shoots were excised and cultured on WPM supplemented with
0.44 μM BA and 0.29 μM GA3. In vitro regenerated shoots were rooted on WPM supplemented with 4.9 μM indole-3-butyric acid. A higher percentage regeneration response
and shoot numbers per explant were recorded on WPM supplemented with BA and GA3, than on MS medium containing BA and TDZ. Lower concentrations of BA and GA3 were required for shoot elongation and prevention of shoot tip necrosis. Each cotyledonary node yielded approximately 20
shoots within 12 wk. Rooted plantlets were successfully acclimatized. 相似文献
13.
Sesbania drummondii (Rydb.) Cory is a source for phytopharmaceuticals. It also hyperaccumulates several toxic heavy metals. Development of an
efficient gene transfer method is an absolute requirement for the genetic improvement of this plant with more desirable traits
due to limitations in conventional breeding methods. A simple protocol was developed for Agrobacterium-mediated stable genetic transformation of Sesbania. Agrobacterium
tumefaciens strain EHA 101 containing the vector pCAMBIA 1305.1 having hptII and GUS plus genes was used for the gene transfer experiments. Evaluation of various parameters was carried out to assess
the transformation frequency by GUS expression analysis. High transformation frequency was achieved by using 7-day-old precultured
cotyledonary node (CN) explants. Further, the presence of acetosyringone (150 μM), infection of explants for 30–45 min and
3 days of cocultivation proved to be critical factors for greatly improving the transformation efficiency. Stable transformation
of S. drummondii was achieved, and putative transgenic shoots were obtained on medium supplemented with hygromycin (25 mg l−1). GUS histochemical analysis of the putative transgenic tissues further confirmed the transformation event. Genomic Southern
blot analysis was performed to verify the presence of transgenes and their stable integration. A transformation frequency
of 4% was achieved for CN explants using this protocol. 相似文献
14.
Meiru Li Hongqing Li Xiaoying Hu Xiaoping Pan Guojiang Wu 《Plant Cell, Tissue and Organ Culture》2011,106(3):363-371
Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol
for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following
co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in
Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l−1 benzyladenine (BA), 0.1 mg l−1 α-naphthaleneacetic acid (NAA), 0.1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l−1 hygromycin, and 500 mg l−1 cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l−1 BA, 0.1 mg l−1 NAA, 0.25 mg l−1 gibberellic acid (GA3), 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots
rooted on MS media supplemented with 0.2 mg l−1 NAA, 20 mg l−1 hygromycin, and 250 mg l−1 cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary
transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic
plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata. 相似文献
15.
Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were
obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT
(2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern
analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants
followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency
as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew
on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization. 相似文献
16.
Sara Rostampour Haleh Hashemi Sohi Esmat Jourabchi Ehsan Ansari 《World journal of microbiology & biotechnology》2009,25(10):1807-1814
Persian poppy (Papaver bracteatum Lindl.) is an important medicinal plant and source of the opium alkaloids codeine, morphine and thebaine. Transgenic root
cultures of P. bracteatum Lindl. are well-defined model systems to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid
biosynthesis. Agrobacterium rhizogenes was able to produce hairy roots on wounded Persian poppy seedlings. Excised shoots from 7-day-old Persian poppy were co-cultivated
with the A. rhizogenes strain R15834 carrying the pBI121 binary vector. All media, except for the co-cultivation medium, included 40 mg l−1 paromomycin to select for pBI121 transformants and 200 mg l−1 cefotaxime to eliminate the Agrobacterium. Eight weeks after infection, paromomycin-resistant roots appeared on 45–50% of explants maintained on hormone-free medium.
Isolated hairy roots were propagated in liquid medium containing 1.0 mg l−1 1-naphthaleneacetic acid to promote rapid growth. Also, callus induction and shoot regeneration of transformed Calli in vitro
was achieved on B5 medium containing 1.0 mg l−1 1-naphthaleneacetic acid. Detection of the neomycin phosphotransferase gene and GUS histochemical localization confirmed
the integrative transformation of root cultures. This is the first study to illustrate useful protocol to introduce foreign
genes into transgenic Persian poppy hairy root cultures using A. rhizogenes strain R15834. 相似文献
17.
A. M. Vieitez E. Corredoira A. Ballester F. Muñoz J. Durán M. Ibarra 《Plant Cell, Tissue and Organ Culture》2009,98(2):135-145
North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal
micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating
and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old
trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot
multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in
explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated
shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture
cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally
placed explants were cultured on media containing 0.2 mg l−1 benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l−1 BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l−1 BA and 0.5 mg l−1 zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO3 (3 mg l−1) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis
and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium
containing 25 mg l−1 indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although
an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets.
However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced
by root, shoot and leaf growth. 相似文献
18.
Elena Palomo-Ríos Araceli Barceló-Muñoz José A. Mercado Fernando Pliego-Alfaro 《Plant Cell, Tissue and Organ Culture》2012,109(2):201-211
Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of
avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for
selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth.
For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant
calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was
confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic
embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar
to control plants. 相似文献
19.
Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring
conservation. In this study, fresh shoot sprouts of P. tuberosa, produced by tubers, were used as explants for in vitro micropropagation. Surface-sterilized nodal shoots were incubated
on Murashige and Skoog (MS) medium supplemented with 8.88 μM benzyladenine (BA), 50 mg l−1 ascorbic acid, and 25 mg l−1 of each of citric acid and adenine sulphate. Cut ends of nodal stem segments rapidly turned brown, and cultures failed to
establish. When 100 mg l−1 ascorbic acid (ABA) and 25.0 mg l−1 polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established. Bud-breaking
of nodal stem explants resulted in multiple shoot formation. Shoots proliferated (35–40 shoots per culture vessel) on MS medium
as described above, but supplemented with 4.44 μM BA and 0.57 μM indole acetic acid (IAA) and additives. After 4–5 passages,
proliferating shoots exhibited tip-browning and decline in growth and multiplication. However, when shoots were transferred
to fresh shoot proliferation medium supplemented with 2.32 μM kinetin (Kn), sustained growth and high rate of shoot proliferation
(50–60 shoots per culture vessel) was observed. Shoots rooted when transferred to medium consisting of half- strength MS medium
with 9.84 μM indole butyric acid (IBA) and 0.02% activated charcoal. Alternatively, individual shoots were pulsed with 984.0 μM
IBA and transferred to glass bottles containing sterile and moistened soilrite. These shoots rooted ex-vitro and were acclimatized
in the greenhouse. Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin. 相似文献
20.
Diane E. Darlington Chiu-Yueh Hung Jiahua Xie 《Plant Cell, Tissue and Organ Culture》2009,99(2):157-165
Agrobacterium
tumefaciens strain LBA4404 containing the plasmid pBI121, carrying the reporter gene uidA and the kanamycin resistance gene nptII, was used for gene transfer experiments in selenium (Se)-hyperaccumulator Astragalus racemosus. The effects of kanamycin on cell growth and division and acetosyringone on transformation efficiency were evaluated. The
optimal concentration of kanamycin that could effectively inhibit cell growth and division in non-transgenic tissues was 50 mg l−1 and thus all putative transgenic plants were obtained on induction medium containing 50 mg l−1 kanamycin. The verification of transformants was achieved by both histochemical GUS assay and PCR amplification of nptII gene. Southern blot analysis was performed to further confirm that transgene nptII was stably integrated into the A. racemosus genome. A transformation frequency of approximately 10% was achieved using this protocol, but no beneficial effect from the
addition of acetosyringone (50 μM) was observed. This transformation system will be a useful tool for future studies of genes
responsible for Se-accumulation in A. racemosus. 相似文献